Enzymatic Resolution by a d-Lactate Oxidase Catalyzed Reaction for (S)-2-Hydroxycarboxylic Acids

Article abstract of DOI:10.1002/cctc.201600536

Oxidase-catalyzed kinetic resolution is important for the production of enantiopure 2-hydroxycarboxylic acids (2-HAs), which are versatile building blocks for the synthesis of many significant compounds. However, in contrast to that of (R)-2-HAs, the production of (S)-2-HA is challenging because of the lack of related oxidases. Herein, suitable enzymes were screened systematically through the analysis of numerous putative d-lactate oxidase sequences and identification of several required properties. Finally, a d-lactate oxidase from Gluconobacter oxydans 621H with advantageous characteristics, such as good solubility, broad substrate spectrum, and high stereoselectivity, was selected to resolve 2-HAs into (S)-2-HAs. A variety of (S)-2-HAs was produced successfully using this d-lactate oxidase with excellent enantiomeric excess values (>99 %). The presented screening criteria and approach for target biocatalysis suggested a guideline for the production of optically active chemicals such as (S)-2-HAs.

Full text of DOI:10.1002/cctc.201600536

DOI: 10.1002/cctc.201600536
Communications
Enzymatic Resolution by a d-Lactate Oxidase Catalyzed
Reaction for (S)-2-Hydroxycarboxylic Acids
Binbin Sheng,[a, b] Jing Xu,[a] Yongsheng Ge,[a] Shuo Zhang,[a] Danqi Wang,[a] Chao Gao,*[a]
Cuiqing Ma,[a] and Ping Xu[c]
Oxidase-catalyzed kinetic resolution is important for the pro-
duction of enantiopure 2-hydroxycarboxylic acids (2-HAs),
which are versatile building blocks for the synthesis of many
significant compounds. However, in contrast to that of
(R)-2-HAs, the production of (S)-2-HA is challenging because of
the lack of related oxidases. Herein, suitable enzymes were
screened systematically through the analysis of numerous pu-
tative d-lactate oxidase sequences and identification of several
required properties. Finally, a d-lactate oxidase from Glucono-
bacter oxydans 621H with advantageous characteristics, such
as good solubility, broad substrate spectrum, and high stereo-
selectivity, was selected to resolve 2-HAs into (S)-2-HAs. A vari-
ety of (S)-2-HAs was produced successfully using this d-lactate
oxidase with excellent enantiomeric excess values (>99%).
The presented screening criteria and approach for target bio-
catalysis suggested a guideline for the production of optically
active chemicals such as (S)-2-HAs.
biocatalyst to resolve racemates to produce optically pure
2-HAs.[10]
A variety of l-2-hydroxy acid oxidases, which include glyco-
late oxidase,[11] l-lactate oxidase,[12] and long-chain l-2-hydroxy
acid oxidase,[13] have been studied intensively. These soluble
enzymes can catalyze the oxidation of (S)-2-HAs directly using
O2 as an electron acceptor. Accordingly, numerous optically
pure (R)-2-HAs have been produced using these l-2-hydroxy
acid oxidases that catalyze the oxidation of (S)-2-HAs in race-
mic 2-HAs.[14] (S)-2-HAs are as important chiral precursors for
the synthesis of diverse functional compounds as (R)-2-HAs.[15]
Skopan et al. and Yamada et al. did pioneering work on the
production of several (S)-2-HAs through an isopropylmalate de-
hydrogenase (which required NAD as a cofactor)[16] and a
Mo-containing 2-oxocarboxylate reductase (which required car-
bamoylmethylviologen as an artificial acceptor).[17]
As in the situation of (R)-2-HA production, it is desirable to
explore the d-2-hydroxy acid oxidases that do not require ex-
pensive cofactors (or artificial electron acceptors) and exhibit
broad substrate spectra for the production of various valuable
(S)-2-HAs. However, there has been no previous report of
(S)-2-HA production from a racemate using an oxidase. In this
study, we intend to introduce a new biocatalytic process to
produce (S)-2-HAs using a d-2-hydroxy acid oxidase in combi-
nation with a catalase (Scheme 1). The target d-2-hydroxy acid
oxidase should possess the following characteristics: (i) good
solubility (a precondition for use on a large scale); (ii) broad
substrate spectrum toward (R)-2-HAs; (iii) the use of O2 as an
electron acceptor; and (iv) high stereoselectivity toward
(R)-isomers.
Optically active 2-hydroxycarboxylic acids (2-HAs) are versatile
building blocks for the asymmetric synthesis of various signifi-
cant compounds.[1] Various methods, which include kinetic res-
olution,[2] chromatographic separation,[3] asymmetric synthe-
sis,[4] and asymmetric reduction,[5] have been explored for use
in the production of chiral 2-HAs. Abundant racemic 2-HAs are
relatively easy to obtain from biological sources and chemical
processes.[6] Hence, kinetic resolution, particularly enzymatic
resolution with high stereoselectivity and mild conditions, is
a promising alternative to prepare enantiopure 2-HAs.[7] Lipase
is the most frequently used biocatalyst for 2-HA resolution.[8]
Oxidase, which requires just O2 as a cosubstrate and no extra
esterification or de-esterification processes,[9] is also a suitable
To date, no d-2-hydroxy acid oxidase with the characteristics
described above has been reported to be used in biocatalysis.
Some enzymes that oxidize d-2-hydroxy acid, which include
[a] Dr. B. Sheng, J. Xu, Y. Ge, S. Zhang, D. Wang, Dr. C. Gao, Prof. C. Ma
State Key Lab of Microbial Technology
Shandong University
27 Shanda South Road, Jinan 250100 (China)
E-mail: jieerbu@sdu.edu.cn
[b] Dr. B. Sheng
School of Environmental Science and Engineering
Sun Yat-sen University
Guangzhou 510275 (China)
[c] Prof. P. Xu
State Key Laboratory of Microbial Metabolism
and School of Life Sciences and Biotechnology
Shanghai Jiao Tong University
800 Dongchuan Road, Shanghai 200240 (China)
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cctc.201600536.
Scheme 1. Enantioselective oxidation of racemic 2-HAs to 2-ketocarboxylic
acids and (S)-2-HAs with O2 catalyzed by putative d-lactate oxidase.
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NAD-independent d-lactate dehydrogenases (d-iLDHs) in-
volved in d-lactate metabolism in vivo, have been character-
ized previously.[18] However, most of these enzymes are mem-
brane-bound, require artificial electron acceptors for the oxida-
tion of d-lactate in vitro, and have narrow substrate spectra,[18]
which makes them unsuitable for use in biocatalysis. The wide
distribution and availability of abundant sequence information
from various sequenced organisms provide an opportunity to
screen a d-iLDH with the desired characteristics for (S)-2-HA
production.
The candidate genes that encoded the putative d-LOXs,
which included SC-LOX (DLD3, protein id=“AJV36019.1”) from
S. cerevisiae, GO-LOX (GOX2071, protein id=“AAW61807.1”)
from G. oxydans 621H, BCa-LOX (protein id=“AEP01915.1”),
BCb-LOX (protein id=“AEP02239.1”) from Bacillus coagulans
36D1, LD-LOX (protein id=“CAI96943.1”) from Lactobacillus
delbrueckii subsp. bulgaricus ATCC 11842, and BC-LOX (protein
id=“AEH53827.1”) from Bacillus coagulans 2–6 (Table S2), were
cloned into the pETDuet-1 vector with an N-terminal His6-tag
and transformed to Escherichia coli BL21 (DE3) for overexpres-
sion. For membrane-bound proteins, a suitable surfactant must
be added for the stability of proteins from the cell membrane
during purification and storage.[18–19] However, sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed
that all seven overexpressed enzymes above could be purified
by using a HisTrap HP column without the addition of any sur-
factant (Figure S2), which indicated that none of the enzymes
are membrane-bound (i.e., they are soluble) in accordance
with the present hypothesis (Figure 1A).
Membrane-bound d-iLDHs are rather difficult to overexpress
and purify on a large scale[18–19] and thus are unsuitable for use
in biocatalysis. The initial screening strategy used here in-
volved solubility as the first criterion. The sequence features of
reported d-iLDHs and their homologous proteins (from >100
genomes) were analyzed in the Pfam database (Table S1 and
Figure S1). The results showed that most proteins contained
a membrane-associated sequence (Figure S1). Actually, only
two soluble d-iLDHs, DLD3 in Saccharomyces cerevisiae[20] and
GOX2071 in Gluconobacter oxydans,[21] have been characterized
previously. Other reported soluble d-iLDHs have been shown
to exist in the crude extracts of lactic acid bacteria (LAB).[22]
Analysis of these two soluble d-iLDHs in the Pfam database in-
dicated that they shared a similar sequence feature, which con-
tained only an FAD-binding domain at the N terminus and an
FAD-oxidase domain at the C terminus. For this reason, several
candidate proteins from LAB with the sequence feature of
soluble d-iLDHs, together with DLD3 and GOX2071, were se-
lected for further screening as possible putative d-lactate
oxidases (d-LOXs; Figure 1A).
Subsequently, the substrate spectra of the purified enzymes
were assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) as an artificial electron acceptor. Ac-
tivity assays were performed at 308C and 578 nm in 0.8 mL of
50 mm Tris-HCl buffer (pH 7.4) that contained 10 mm various
2-HAs, 0.2 mm MTT, 0.2 mm phenazine methosulfate (PMS),
and the purified enzymes. No detectable activities were ob-
served in the enzyme solution of BCa-LOX, LD-LOX, and
BC-LOX, although the purified soluble enzymes were produced
in large quantities. In contrast, purified SC-LOX, GO-LOX, and
BCb-LOX showed activities and preferences toward various
2-HAs (Table S3). These three enzymes with broad substrate
spectra were subjected to subsequent screening
experiments (Figure 1A).
To further assess the catalytic capacity of SC-LOX,
GO-LOX, and BCb-LOX using O2 as an electron ac-
ceptor, their specific activities for various 2-HAs (1a–
16a) were assayed by using a Clark-type oxygen
electrode with 10 mm 2-HAs and an appropriate
amount of enzyme solution in 50 mm air-saturated
Tris-HCl buffer (pH 7.4) at 900 rpm and 308C. These
three enzymes all showed catalytic capacity directly
using O2 (Table 1). Although their specific activities
toward different electron acceptors (MTT and O2)
were not equivalent, all enzymes showed similar
trends in their catalytic properties. For various 2-HAs
with different side-chain lengths, SC-LOX showed
the greatest activity toward short aliphatic chains,
BCb-LOX preferred longer aliphatic or aromatic
chains, and GO-LOX preferred nearly all (R)-2-HAs
with aliphatic and aromatic chains. GO-LOX also
showed high oxidase activities toward most 2-HAs
that contained substituent groups (10a–16a). With
regard to stereoselectivity, GO-LOX also performed
better than SC-LOX and BCb-LOX. In this way, after
sequential screening (Figure 1A), GO-LOX was select-
ed as the most appropriate enzyme for use in the re-
action shown in Scheme 1. Then the enzymatic
Figure 1. A) Screening process for suitable d-LOX and B,C) its application for the produc-
tion of (S)-2a, and (S)-7a. &, 2-HA; *, 2-ketocarboxylic acid; ~, ee.
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GO-LOX (0.5 or 1 mgmLÀ1 according to its activities toward the
various substrates), and 0.05 mgmLÀ1 catalase for 12 h at 308C
(Table 2). As GO-LOX had a very low O2-oxidant activity toward
(R)-6a, 10a, 12a, and 15a, a halved substrate concentration
(10 mm) and a long reaction time (24 h) were used. All racemic
1a–16a (including 6a, 10a, 12a, and 15a) were resolved suc-
cessfully into S isomers with excellent enantiomeric excess (ee)
values (>99%; Table 2 and Figure S4) and high conversions
(ꢀ50%). The corresponding 2-ketocarboxylic acids, another
type of valuable chemicals, were also produced.
Table 1. Comparison of the specific activity of putative d-LOXs for 2-HAs
with O2 as an electron acceptor.
Entry
Substrate
Specific activity [U mgÀ1
GO-LOX
]
SC-LOX
BCb-LOX
1
2
3
4
5
6
7
8
(R)-1a
(S)-1a
(R)-2a
(S)-2a
rac-3a
rac-4a
rac-5a
rac-6a
(R)-7a
(S)-7a
(R)-8a
(S)-8a
(R)-9a
0.47
0.27
0.49
0.02
0.35
0.08
0.05
0.04
0.00
0.00
0.00
0.00
0.01
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.40
0.00
0.24
0.00
0.09
0.08
0.13
0.01
0.31
0.00
0.17
0.00
0.15
0.00
0.03
0.22
0.07
0.35
0.26
0.05
0.20
0.04
0.02
0.04
0.01
0.05
0.02
0.10
0.23
0.16
0.00
0.01
0.00
0.19
0.02
0.01
0.04
0.01
0.04
0.04
0.01
0.01
To resolve racemic 2-HAs into (S)-2-HAs using oxidase, suita-
ble enzymes were screened systematically by analyzing numer-
ous putative d-LOX sequences and sequentially identifying sev-
eral required properties, which included solubility, substrate
spectrum, oxidase activity, and stereoselectivity (Figure 1A).
GO-LOX was found to be the best biocatalyst for the kinetic
resolution of 2-HAs (Figure 1A). To further assess the actual po-
tential of GO-LOX and to monitor the reaction processes, two
amplified reactions were performed to resolve racemic 2a and
7a (aliphatic and aromatic, respectively) using an elevated sub-
strate concentration (100 mm) and an amplified reaction
volume (5 mL) from which samples were withdrawn at inter-
vals and analyzed by using HPLC. As expected, (S)-2a and
(S)-7a were produced with excellent ee values (>99%) within
60 h, together with the high production of 2b and 7b (Fig-
ure 1B and C). In addition, given the high production of 2b
and 7b, an enantioselective two-step stereoinversion[23] of
rac-a to (S)-a can be further investigated to improve the theo-
retical conversion of the reaction from 50 to 100%. Taking 2a
as an example, the conversion of the reaction to produce
(S)-2a from 100 mm rac-2a reached up to 100% with excellent
ee values (>99%; Figure S5) by coupling a high stereoselectiv-
ity NAD-dependent l-lactate dehydrogenase (l-nLDH)[24] to the
kinetic resolution reaction, as expected.
9
10
11
12
13
14
15
16
17
18
19
20
21
(S)-9a
rac-10a
rac-11 a
rac-12a
rac-13a
rac-14a
rac-15a
rac-16a
properties of GO-LOX were assessed. Its optimal temperature
is 558C and it is stable below 358C. Its optimal pH is 8 and it is
stable in a buffer of pH 7–9 (Figure S3).
To assess the applicability of GO-LOX for the kinetic resolu-
tion of 2-HAs, the reaction solutions were prepared and react-
ed in 1 mL of 50 mm Tris-HCl buffer (pH 7.4) that contained
20 mm racemic substrates (except for 6a, 10a, 12a, and 15a),
Table 2. Kinetic resolution of racemic 2-HAs with GO-LOX.[a]
In summary, we used a d-lactate oxidase (GO-LOX) found by
systematic screening as the biocatalyst to develop a promising
approach for the production of various (S)-2-HAs, (S)-1a–16a,
by kinetic resolution with excellent ee values (>99%) and high
conversions. The screening criteria and processes for the target
biocatalyst in this study also provide a guideline for the pro-
duction of high-value enantiopure chemicals such as
(S)-2-HAs.
Substrate
[mM]
GO-LOX
t
[h]
Conversion
[%][b]
eeS
Product b
[mgmLÀ1
]
[%][c]
[mM][d]
1a (20)
2a (20)
3a (20)
4a (20)
5a (20)
6a (10)
7a (20)
8a (20)
9a (20)
10a (10)
11 a (20)
12a (10)
13a (20)
14a (20)
15a (10)
16a (20)
0.5
0.5
1
1
1
1
0.5
1
1
1
1
1
1
1
1
1
12
12
12
12
12
24
12
12
12
24
12
24
12
12
24
12
51.5
50
50
51
55
51
51.5
50
50
50
50
50
50
50
50
50
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
10.3
9.8
9.2
9.0
11.0
5.1
10.3
9.8
9.6
~5
~10
~5
Acknowledgements
This study was supported by the National Natural Science Foun-
dation of China (31270856 and 31170052), the Chinese National
Program for High Technology Research and Development
(2014AA021206), and the Young Scholars Program of Shandong
University (2015WLJH25). We thank Prof. Xiaoming Bao (Shan-
dong University, China) for providing a genome sample of S. cer-
evisiae CEN.PK 102-3A.
~10
~10
~5
~10
[a] Enzymatic resolution reactions were performed at 308C in 1 mL of
50 mm Tris-HCl buffer (pH 7.4) that contained racemic substrates,
GO-LOX, and 0.05 mgmLÀ1 catalase. Samples were determined by using
HPLC. [b] Conversion was calculated as follows: [(the initial rac-aÀthe re-
sidual a)/the initial rac-a]ꢁ100%. [c] The ee values of (S)-a were defined
as follows: [((S)-aÀ(R)-a)/((S)-a+(R)-a)]ꢁ100%. [d] As a result of the lack of
10b–16b standards, they were analyzed qualitatively by using LC–MS
(Figure S4).
Keywords: carboxylic acids
catalysis · kinetic resolution
· enantioselectivity · enzyme
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Received: May 3, 2016
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B. Sheng, J. Xu, Y. Ge, S. Zhang, D. Wang,
C. Gao,* C. Ma, P. Xu
&& – &&
Enzymatic Resolution by a d-Lactate
Oxidase Catalyzed Reaction for (S)-2-
Hydroxycarboxylic Acids
Enzymatic resolution: d-Lactate
carboxylic acids, which are important
platform compounds. A variety of (S)-2-
hydroxycarboxylic acids (16 examples)
was produced using this d-lactate ox-
idase with excellent enantiomeric
excess values (>99%).
oxidase, which has advantageous char-
acteristics of good solubility, a broad
substrate spectrum, and high stereose-
lectivity, was selected to resolve 2-hy-
droxycarboxylic acids into (S)-2-hydroxy-
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